Methods for the treatment of cachexia and graft v. host disease

ABSTRACT

Substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and 1-oxo-2-(2,6-dioxopiperidin-3-yl)isoindolines are disclosed. The compounds are useful, for example, in reducing the levels of TNFα in a mammal.

This is a division of co-pending U.S. application Ser. No. 10/337,602,filed Jan. 6, 2003 now U.S. Pat. No. 7,119,106, which is a continuationof U.S. application Ser. No. 09/781,179, filed Feb. 12, 2001, now U.S.Pat. No. 6,555,554, which is a continuation of U.S. application Ser. No.09/543,809, filed Apr. 6, 2000, now U.S. Pat. No. 6,281,230, which is adivision of U.S. application Ser. No. 09/230,389, filed May 7, 1999, nowabandoned, which is based on application no. PCT/US97/13375, filed Jul.24, 1997, which claims the benefit of U.S. application Ser. No.08/690,258, filed Jul. 24, 1996, now U.S. Pat. No. 5,635,517 and Ser.No. 08/701,494, filed Aug. 22, 1996, now U.S. Pat. No. 5,798,368, and ofU.S. provisional application No. 60/048,278, filed May 30, 1997, all ofwhich are incorporated herein in their entireties by reference.

The present invention relates to substituted2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines, the method of reducinglevels of tumor necrosis factor α in a mammal through the administrationthereof, and pharmaceutical compositions of such derivatives.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α, or TNFα, is a cytokine which is releasedprimarily by mononuclear phagocytes in response to a numberimmunostimulators. When administered to animals or humans, it causesinflammation, fever, cardiovascular effects, hemorrhage, coagulation,and acute phase responses similar to those seen during acute infectionsand shock states. Excessive or unregulated TNFα production thus has beenimplicated in a number of disease conditions. These include endotoxemiaand/or toxic shock syndrome {Tracey et al., Nature 330, 662-664 (1987)and Hinshaw et al., Circ. Shock 30, 279-292 (1990)}; cachexia {Dezube etal., Lancet, 335 (8690), 662 (1990)} and Adult Respiratory DistressSyndrome where TNFα concentration in excess of 12,000 pg/mL have beendetected in pulmonary aspirates from ARDS patients {Millar et al.,Lancet 2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNFαalso resulted in changes typically seen in ARDS {Ferrai-Baliviera etal., Arch. Surg. 124(12), 1400-1405 (1989)}.

TNFα appears to be involved in bone resorption diseases, includingarthritis. When activated, leukocytes will produce bone-resorption, anactivity to which the data suggest TNFα contributes. {Bertolini el al.,Nature 319, 516-518 (1986) and Johnson et al., Endocrinology 124(3),1424-1427 (1989).} TNFα also has been shown to stimulate bone resorptionand inhibit bone formation in vitro and in vivo through stimulation ofosteoclast formation and activation combined with inhibition ofosteoblast function. Although TNFα may be involved in many boneresorption diseases, including arthritis, the most compelling link withdisease is the association between production of TNFα by tumor or hosttissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US)46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increasedserum TNFα levels have been associated with major complication followingacute allogenic bone marrow transplants {Holler et al., Blood, 75(4),1011-1016 (1990)}.

Cerebral malaria is a lethal hyperacute neurological syndrome associatedwith high blood levels of TNFα and the most severe complicationoccurring in malaria patients. Levels of serum TNFα correlated directlywith the severity of disease and the prognosis in patients with acutemalaria attacks {Grau et al., N. Engl. J. Med. 320(24), 1586-1591(1989)}.

Macrophage-induced angiogenesis TNFα is known to be mediated by TNFα.Leibovich et al. {Nature, 329, 630-632 (1987)} showed TNFα induces invivo capillary blood vessel formation in the rat cornea and thedeveloping chick chorioallantoic membranes at very low doses and suggestTNFα is a candidate for inducing angiogenesis in inflammation, woundrepair, and tumor growth. TNFα production also has been associated withcancerous conditions, particularly induced tumors {Ching et al, Brit. J.Cancer, (1955) 72, 339-343, and Koch, Progress in Medicinal Chemistry,22, 166-242 (1985)}.

TNFα also plays a role in the area of chronic pulmonary inflammatorydiseases. The deposition of silica particles leads to silicosis, adisease of progressive respiratory failure caused by a fibroticreaction. Antibody to TNFα completely blocked the silica-induced lungfibrosis in mice {Pignet et al., Nature, 344:245-247 (1990)}. Highlevels of TNFα production (in the serum and in isolated macrophages)have been demonstrated in animal models of silica and asbestos inducedfibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}.Alveolar macrophages from pulmonary sarcoidosis patients have also beenfound to spontaneously release massive quantities of TNFα as comparedwith macrophages from normal donors {Baughman et al., J. Lab. Clin. Med.115(1), 36-42 (1990)}.

TNFα is also implicated in the inflammatory response which followsreperfusion, called reperfusion injury, and is a major cause of tissuedamage after loss of blood flow {Vedder et al., PNAS 87, 2643-2646(1990)}. TNFα also alters the properties of endothelial cells and hasvarious pro-coagulant activities, such as producing an increase intissue factor pro-coagulant activity and suppression of theanticoagulant protein C pathway as well as down-regulating theexpression of thrombomodulin {Sherry et al., J. Cell Biol. 107,1269-1277 (1988)}. TNFα has pro-inflammatory activities which togetherwith its early production (during the initial stage of an inflammatoryevent) make it a likely mediator of tissue injury in several importantdisorders including but not limited to, myocardial infarction, strokeand circulatory shock. Of specific importance may be TNFα-inducedexpression of adhesion molecules, such as intercellular adhesionmolecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) onendothelial cells {Munro et al., Am. J. Path. 135(1), 121-132 (1989)}.

TNFα blockage with monoclonal anti-TNFα antibodies has been shown to bebeneficial in rheumatoid arthritis {Elliot et al., Int. J. Pharmac. 199517(2), 141-145} and Crohn's disease {von Dullemen et al.,Gastroenterology, 1995 109(1), 129-135}

Moreover, it now is known that TNFα is a potent activator of retrovirusreplication including activation of HIV-1. {Duh et al., Proc. Nat. Acad.Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87,782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J.Immunol. 142, 431438 (1989); Poll et al., AIDS Res. Hum. Retrovirus,191-197 (1992)}. AIDS results from the infection of T lymphocytes withHuman Immunodeficiency Virus (HIV). At least three types or strains ofHIV have been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequenceof HIV infection, T-cell mediated immunity is impaired and infectedindividuals manifest severe opportunistic infections and/or unusualneoplasms. HIV entry into the T lymphocyte requires T lymphocyteactivation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytesafter T cell activation and such virus protein expression and/orreplication is mediated or maintained by such T cell activation. Once anactivated T lymphocyte is infected with HIV, the T lymphocyte mustcontinue to be maintained in an activated state to permit HIV geneexpression and/or HIV replication. Cytokines, specifically TNFα, areimplicated in activated T-cell mediated HIV protein expression and/orvirus replication by playing a role in maintaining T lymphocyteactivation. Therefore, interference with cytokine activity such as byprevention or inhibition of cytokine production, notably TNFα, in anHIV-infected individual assists in limiting the maintenance of Tlymphocyte caused by HIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glialcells, also have been implicated in maintenance of the HIV infection.These cells, like T cells, are targets for viral replication and thelevel of viral replication is dependent upon the activation state of thecells. {Rosenberg et al., The Immunopathogenesis of HIV Infection,Advances in Immunology, 57 (1989)}. Cytokines, such as TNFα, have beenshown to activate HIV replication in monocytes and/or macrophages {Poliet al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)}, therefore,prevention or inhibition of cytokine production or activity aids inlimiting, HIV progression for T cells. Additional studies haveidentified TNFα as a common factor in the activation of HIV in vitro andhas provided a clear mechanism of action via a nuclear regulatoryprotein found in the cytoplasm of cells (Osborn, et al., PNAS 862336-2340). This evidence suggests that a reduction of TNFα synthesismay have an antiviral effect in HIV infections, by reducing thetranscription and thus virus production.

AIDS viral replication of latent HIV in T cell and macrophage lines canbe induced by TNFα {Folks et al., PNAS 86, 2365-2368 (1989)}. Amolecular mechanism for the virus inducing activity is suggested byTNFα's ability to activate a gene regulatory protein (NFκB) found in thecytoplasm of cells, which promotes HIV replication through binding to aviral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340(1989)}. TNFα in AIDS associated cachexia is suggested by elevated serumTNFα and high levels of spontaneous TNFα production in peripheral bloodmonocytes from patients {Wright et al., J. Immunol. 141(1), 99-104(1988)}. TNFα has been implicated in various roles with other viralinfections, such as the cytomegalia virus (CMV), influenza virus,adenovirus, and the herpes family of viruses for similar reasons asthose noted.

The nuclear factor κB (NFκB) is a pleiotropic transcriptional activator(Lenardo, et al., Cell 1989, 58, 227-29). NFκB has been implicated as atranscriptional activator in a variety of disease and inflammatorystates and is thought to regulate cytokine levels including but notlimited to TNFα and also to be an activator of HIV transcription(Dbaibo, et al., J. Biol. Chem. 1993, 17762-66; Duh et al., Proc. Natl.Acad Sci. 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350,709-12; Boswas et al., J. Acquired Immune Deficiency Syndrome 1993, 6,778-786; Suzuki et al., Biochem. And Biophys. Res. Comm. 1993, 193,277-83; Suzuki et al., Biochem. And Biophys. Res Comm. 1992, 189,1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700;Shakhov et al., Proc. Natl. Acad. Sci. USA 1990, 171, 35-47; and Staalet, al., Proc. Natl. Acad sci. USA 1990, 87, 9943-47). Thus, inhibitionof NFκB binding can regulate transcription of cytokine gene(s) andthrough this modulation and other mechanisms be useful in the inhibitionof a multitude of disease states. The compounds described herein caninhibit the action of NFκB in the nucleus and thus are useful in thetreatment of a variety of diseases including but not limited torheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, otherarthritic conditions, septic shock, septis, endotoxic shock, graftversus host disease, wasting, Crohn's disease, ulcerative colitis,multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV,AIDS, and opportunistic infections in AIDS. TNFα and NFκB levels areinfluenced by a reciprocal feedback loop. As noted above, the compoundsof the present invention affect the levels of both TNFα and NFκB.

Many cellular functions are mediated by levels of adenosine 3′,5′-cyclicmonophosphate (cAMP). Such cellular functions can contribute toinflammatory conditions and diseases including asthma, inflammation, andother conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807,1992). It has been shown that the elevation of cAMP in inflammatoryleukocytes inhibits their activation and the subsequent release ofinflammatory mediators, including TNFα and NFκB. Increased levels ofcAMP also leads to the relaxation of airway smooth muscle.

Decreasing TNFα levels and/or increasing cAMP levels thus constitutes avaluable therapeutic strategy for the treatment of many inflammatory,infectious, immunological, and malignant diseases. These include but arenot restricted to septic shock, sepsis, endotoxic shock, hemodynamicshock and sepsis syndrome, post ischemic reperfusion injury, malaria,mycobacterial infection, meningitis, psoriasis, congestive heartfailure, fibrotic disease, cachexia, graft rejection, oncogenic orcancerous conditions, asthma, autoimmune disease, opportunisticinfections in AIDS, rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, other arthritic conditions, Crohn's disease, ulcerativecolitis, multiple sclerosis, systemic lupus erythrematosis, ENL inleprosy, radiation damage, oncogenic conditions, and hyperoxic alveolarinjury. Prior efforts directed to the suppression of the effects of TNFαhave ranged from the utilization of steroids such as dexamethasone andprednisolone to the use of both polyclonal and monoclonal antibodies{Beutler et al., Science 234, 470474 (1985); WO 92/11383}.

DETAILED DESCRIPTION

The present invention is based on the discovery that certain classes ofnon-polypeptide compounds more fully described herein decrease thelevels of TNFα.

In particular, the invention pertains to (i) compounds of the formula:

-   -   in which:        -   one of X and Y is C═O and the other of X and Y is C═O or            CH₂;        -   (i) each of R¹, R², R³, and R⁴, independently of the others,            is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4            carbon atoms or (ii) one of R¹, R², R³, and R⁴ is —NHR⁵ and            the remaining of R¹, R², R³, and R⁴ are hydrogen;        -   R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;        -   R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or            halo;        -   provided that R⁶ is other than hydrogen if X and Y are C═O            and (i) each of R¹, R², R³, and R⁴ is fluoro or (ii) one of            R¹, R², R³, or R⁴ is amino; and    -   (b) the acid addition salts of said compounds which contain a        nitrogen atom capable of being protonated.

A preferred group of compounds are those of Formula I in which each ofR¹, R², R³, and R⁴, independently of the others, is halo, alkyl of 1 to4 carbon atoms, or alkoxy of 1 to 4 carbon atoms, and R⁶ is hydrogen,methyl, ethyl, or propyl. A second preferred group of compounds arethose of Formula I in which one of R¹, R², R³, and R⁴ is —NH₂, theremaining of R¹, R², R³, and R⁴ are hydrogen, and R⁶ is hydrogen,methyl, ethyl, or propyl.

Unless otherwise defined, the term alkyl denotes a univalent saturatedbranched or straight hydrocarbon chain containing from 1 to 8 carbonatoms. Representative of such alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. Alkoxy refers toan alkyl group bound to the remainder of the molecule through anethereal oxygen atom. Representative of such alkoxy groups are methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, andtert-butoxy. Preferably R¹, R², R³, and R⁴ are chloro, fluoro, methyl ormethoxy.

The compounds of Formula I are used, under the supervision of qualifiedprofessionals, to inhibit the undesirable effects of TNFα. The compoundscan be administered orally, rectally, or parenterally, alone or incombination with other therapeutic agents including antibiotics,steroids, etc., to a mammal in need of treatment.

The compounds of the present invention also can be used topically in thetreatment or prophylaxis of topical disease states mediated orexacerbated by excessive TNFα production, respectively, such as viralinfections, such as those caused by the herpes viruses, or viralconjunctivitis, psoriasis, atopic dermatitis, etc.

The compounds also can be used in the veterinary treatment of mammalsother than humans in need of prevention or inhibition of TNFαproduction. TNFα mediated diseases for treatment, therapeutically orprophylactically, in animals include disease states such as those notedabove, but in particular viral infections. Examples include felineimmunodeficiency virus, equine infectious anraemia virus, caprinearthritis virus, visna virus, and maedi virus, as well as otherlentiviruses.

Compounds in which one of R¹, R², R³, R⁴ is amino and R⁵ and R⁶, as wellas the remainder of R¹, R², R³, R⁴, are hydrogen as for example,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline or1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline are known. See,e.g., Jönsson, Acta Pharma. Succica, 9, 521-542 (1972).

The compounds can be prepared using methods which are known in general.In particular, the compounds can be prepared through the reaction of2,6-dioxopiperidin-3-ammonium chloride, and a lower alkyl ester of2-bromomethylbenzoic acid in the presence of an acid acceptor such asdimethylaminopyridine or triethyl amine.

The substituted benzoate intermediates are known or can be obtainedthough conventional processes. For example, a lower alkyl ester of anortho-toluic acid is brominated with N-bromosuccinimide under theinfluence of light to yield the lower alkyl 2-bromomethylbenzoate.

Alternatively, a dialdehyde is allowed to react with2,6-dioxopiperidin-3-ammonium chloride:

In a further method, a dialdehyde is allowed to react with glutamine andthe resulting 2-(1-oxoisoindolin-2-yl)glutaric acid then cyclized toyield a 1-oxo-2-(2,6-dioxopiperidin-3-yl)-isoindoline of Formula I:

Finally, an appropriately substituted phthalidimide intermediate isselectively reduced:

Amino compounds can be prepared through catalytic hydrogenation of thecorresponding nitro compound:

The nitro intermediates of Formula IA are known or can be obtainedthough conventional processes. For example, a nitrophthalic anhydride isallowed to react with α-aminoglutarimide hydrochloride {alternativelynamed as 2,6-dioxopiperidin-3-ylammonium chloride} in the presence ofsodium acetate and glacial acetic acid to yield an intermediate ofFormula IA in which X and Y are both C═O.

In a second route, a lower alkyl ester of nitro-ortho-toluic acid isbrominated with N-bromosuccinimide under the influence of light to yielda lower alkyl 2-(bromomethyl)nitrobenzoate. This is allowed to reactwith 2,6-dioxopiperidin-3-ammonium chloride in, for example,dimethylformamide in the presence of triethylamine to yield anintermediate of Formula II in which one of X is C═O and the other isCH₂.

Alternatively, if one of R¹, R₂, R₃, and R₄ is protected amino, theprotecting group can be cleaved to yield the corresponding compound inwhich one of R¹, R₂, R₃, and R⁴ is amino. Protecting groups utilizedherein denote groups which generally are not found in the finaltherapeutic compounds but which are intentionally introduced at somestage of the synthesis in order to protect groups which otherwise mightbe altered in the course of chemical manipulations. Such protectinggroups are removed at a later stage of the synthesis and compoundsbearing such protecting groups thus are of importance primarily aschemical intermediates (although some derivatives also exhibitbiological activity). Accordingly the precise structure of theprotecting group is not critical. Numerous reactions for the formationand removal of such protecting groups are described in a number ofstandard works including, for example, “Protective Groups in OrganicChemistry”, Plenum Press, London and New York, 1973; Greene, Th. W.“Protective Groups in Organic Synthesis”, Wiley, New York, 1981; “ThePeptides”, Vol. I, Schröder and Lubke, Academic Press, London and NewYork, 1965; “Methoden der organischen Chemie”, Houben-Weyl, 4th Edition,Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974, the disclosures of whichare incorporated herein by reference. An amino group can be protected asan amide utilizing an acyl group which is selectively removable undermild conditions, especially benzyloxycarbonyl, formyl, or a loweralkanoyl group which is branched in 1- or α position to the carbonylgroup, particularly tertiary alkanoyl such as pivaloyl, a lower alkanoylgroup which is substituted in the position a to the carbonyl group, asfor example trifluoroacetyl.

The compounds of the present invention possess a center of chirality andcan exist as optical isomers. Both the racemates of these isomers andthe individual isomers themselves, as well as diastereomers when thereare two chiral centers, are within the scope of the present invention.The racemates can be used as such or can be separated into theirindividual isomers mechanically as by chromatography using a chiraladsorbent. Alternatively, the individual isomers can be prepared inchiral form or separated chemically from a mixture by forming salts witha chiral acid, such as the individual enantiomers of 10-camphorsulfonicacid, camphoric acid, α-bromocamphoric acid, methoxyacetic acid,tartaric acid, diacetyltartaric acid, malic acid,pyrrolidone-5-carboxylic acid, and the like, and then freeing one orboth of the resolved bases, optionally repeating the process, so asobtain either or both substantially free of the other; i.e., in a formhaving an optical purity of >95%.

The present invention also pertains to the physiologically acceptablenon-toxic acid addition salts of the compounds of Formula I. Such saltsinclude those derived from organic and inorganic acids such as, withoutlimitation, hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid, methanestulphonic acid, acetic acid, tartaric acid,lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbicacid, aconitic acid, salicylic acid, phthalic acid, embonic acid,enanthic acid, and the like.

Oral dosage forms include tablets, capsules, dragees, and similarshaped, compressed pharmaceutical forms containing from 1 to 100 mg ofdrug per unit dosage. Isotonic saline solutions containing from 20 to100 mg/mL can be used for parenteral administration which includesintramuscular, intrathecal, intravenous and intra-arterial routes ofadministration. Rectal administration can be effected through the use ofsuppositories formulated from conventional carriers such as cocoabutter.

Pharmaceutical compositions thus comprise one or more compounds of thepresent invention associated with at least one pharmaceuticallyacceptable carrier, diluent or excipient. In preparing suchcompositions, the active ingredients are usually mixed with or dilutedby an excipient or enclosed within such a carrier which can be in theform of a capsule or sachet. When the excipient serves as a diluent, itmay be a solid, semi-solid, or liquid material which acts as a vehicle,carrier, or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, elixirs, suspensions,emulsions, solutions, syrups, soft and hard gelatin capsules,suppositories, sterile injectable solutions and sterile packagedpowders. Examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidinone, cellulose, water,syrup, and methyl cellulose, the formulations can additionally includelubricating agents such as talc, magnesium stearate and mineral oil,wetting agents, emulsifying and suspending agents, preserving agentssuch as methyl- and propylhydroxybenzoates, sweetening agents orflavoring agents.

The compositions preferably are formulated in unit dosage form, meaningphysically discrete units suitable as a unitary dosage, or apredetermined fraction of a unitary dose to be administered in a singleor multiple dosage regimen to human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with a suitablepharmaceutical excipient. The compositions can be formulated so as toprovide an immediate, sustained or delayed release of active ingredientafter administration to the patient by employing procedures well knownin the art.

Oral dosage forms include tablets, capsules, dragees, and similarshaped, compressed pharmaceutical forms containing from 1 to 100 mg ofdrug per unit dosage. Isotonic saline solutions containing from 20 to100 mg/mL can be used for parenteral administration which includesintramuscular, intrathecal, intravenous and intra-arterial routes ofadministration. Rectal administration can be effected through the use ofsuppositories formulated from conventional carriers such as cocoabutter.

Pharmaceutical compositions thus comprise one or more compounds of thepresent invention associated with at least one pharmaceuticallyacceptable carrier, diluent or excipient. In preparing suchcompositions, the active ingredients are usually mixed with or dilutedby an excipient or enclosed within such a carrier which can be in theform of a capsule or sachet. When the excipient serves as a diluent, itmay be a solid, semi-solid, or liquid material which acts as a vehicle,carrier, or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, elixirs, suspensions,emulsions, solutions, syrups, soft and hard gelatin capsules,suppositories, sterile injectable solutions and sterile packagedpowders. Examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidinone, cellulose, water,syrup, and methyl cellulose, the formulations can additionally includelubricating agents such as talc, magnesium stearate and mineral oil,wetting agents, emulsifying and suspending agents, preserving agentssuch as methyl- and propylhydroxybenzoates, sweetening agents orflavoring agents.

The compositions preferably are formulated in unit dosage form, meaningphysically discrete units suitable as a unitary dosage, or apredetermined fraction of a unitary dose to be administered in a singleor multiple, dosage regimen to human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with a suitablepharmaceutical excipient.

The compositions can be formulated so as to provide an immediate,sustained or delayed release of active ingredient after administrationto the patient by employing procedures well known in the art.

The following examples will serve to further typify the nature of thisinvention but should not be construed as a limitation in the scopethereof, which scope is defined solely by the appended claims.

Example 1 1,3-Dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline

A mixture of 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-nitroisoindoline{alternatively named as N-(2,6-dioxopiperidin-3-yl)-4-nitrophthalimide}(1 g, 3.3 mmol) and 10% Pd/C (0.13 g) in 1,4-dioxane (200 mL) washydrogenated at 50 psi for 6.5 hours. The catalyst was filtered throughCelite and the filtrate concentrated in vacuo. The residue wascrystallized from ethyl acetate (20 mL) to give 0.62 g (69%) of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline {alternativelynamed as N-(2,6-dioxopiperidin-3-yl)4-aminophthalimide} as an orangesolid. Recrystallization from dioxane/ethyl acetate gave 0.32 g ofyellow solid: mp 318.5-320.5° C.; HPLC (nova Pak C18, 15/85acetonitrile/0.1% H₃PO₄) 3.97 min (98.22%): ¹H NMR (DMSO-d₆) δ 11.08 (s,1H), 7.53-7.50 (d, J=8.3 Hz, 1H), 6.94 (s, 1H), 6.84-6.81 (d, J=8.3 Hz,1H), 6.55 (s, 2H). 5.05-4.98 (m, 1H), 2.87-1.99 (m, 4H); ¹³C NMR(DMSO-d₆) δ 172.79, 170.16, 167.65, 167.14, 155.23, 134.21, 125.22,116.92, 116.17, 107.05, 48.58, 30.97, 22.22; Anal. Calcd for C₁₃H₁₁N₃O₄:C, 57.14; H, 4.06; N, 15.38. Found: C, 56.52; H, 4.17; N, 14.60.

In a similar fashion from1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-nitroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-nitroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-nitroisoindoline, and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline, there isrespectively obtained1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline, and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)4-aminoisoindoline, respectively,upon hydrogenation.

Example 2 1,3-Dioxo-2-(2,6-dioxopiperidin-3-yl)-5-nitroisoindoline

A mixture of 4-nitrophthalic anhydride (1.7 g, 8.5 mmol),α-aminoglutarimide hydrochloride (1.4 g, 8.5 mmol) and sodium acetate(0.7 g, 8.6 mmol) in glacial acetic acid (30 mL) was heated under refluxfor 17 hours. The mixture was concentrated in vacuo and the residue wasstirred with methylene chloride (40 mL) and water (30 mL). The aqueouslayer was separated, extracted with methylene chloride (2×40 mL). Thecombined methylene chloride solutions were dried over magnesium sulfateand concentrated in vacuo to give 1.4 g (54%) of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-nitroisoindoline as a lightbrown solid. An analytical sample was obtained by recrystallization frommethanol: mp 228.5-229.5° C.; ¹H NMR (DMSO-d₆) δ 11.18 (s, 1 H),8.69-8.65 (d, d J=1.9 and 8.0 Hz, 1H), 8.56 (d, J=1.9 Hz, 1H), 8.21 (d,H=8.2 Hz, 1H), 5.28 (d, d J=5.3 and 12.8 Hz, 1H), 2.93-2.07 (m, 4H); ¹³CNMR (DMSO-d₆) δ 172.66, 169.47, 165.50, 165.23, 151.69, 135.70, 132.50,130.05, 124.97, 118.34, 49.46, 30.85, 21.79; Anal. Calcd for C₁₃H₉N₃O₆:C, 51.49; H, 2.99; N, 13.86. Found: C, 51.59; H, 3.07; N, 13.73.

1-Oxo-2-(2,6-dioxopiperidin-3-yl)-5-nitroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-nitroisoindoline, and1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-nitroisoindoline can be obtained byallowing 2,6-dioxopiperidin-3-ammonium chloride to react with methyl2-bromomethyl-5-nitrobenzoate, methyl 2-bromomethyl-4-nitrobenzoate,methyl 2-bromomethyl-6-nitrobenzoate, and methyl2-bromomethyl-7-nitrobenzoate, respectively, in dimethylformamide in thepresence of triethylamine. The methyl 2-(bromomethyl)nitrobenzoates inturn are obtained from the corresponding methyl esters ofnitro-ortho-toluic acids by conventional bromination withN-bromosuccinimide under the influence of light.

Example 31-Oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetranfuoroisoindoline

A mixture of 16.25 g of 2,6-dioxopiperidin-3-ammonium chloride, and 30.1g of methyl 2-bromomethyl-3,4,5,6-tetrafluorobenzoate, and 12.5 g oftriethylamine in 100 mL of dimethylformamide is stirred at roomtemperature for 15 hours. The mixture is then concentrated in vacuo andthe residue mixed with methylene chloride and water. The aqueous layeris separated and back-extracted with methylene chloride. The combinedmethylene chloride solutions are dried over magnesium sulfate andconcentrated in vacuo to give1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline.

In a similar fashion1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline, and1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline areobtained by substituting equivalent amounts of2-bromomethyl-3,4,5,6-tetrachlorobenzoate,2-bromomethyl-3,4,5,6-tetramethylbenzoate, and2-bromomethyl-3,4,5,6-tetramethoxybenzoate, respectively, for2-bromomethyl-3,4,5,6-tetrafluorobenzoate.

Example 4 N-Benzyloxycarbonyl-α-methyl-glutamic Acid

To a stirred solution of α-methyl-D,L-glutamic acid (10 g, 62 mmol) in 2N sodium hydroxide (62 mL) at 0-5° C. was added benzyl chloroformate(12.7 g, 74.4 mmol) over 30 min. After the addition was complete thereaction mixture was stirred at room temperature for 3 hours. Duringthis time the pH was maintained at 11 by addition of 2N sodium hydroxide(33 mL). The reaction mixture was then extracted with ether (60 mL). Theaqueous layer was cooled in an ice bath and then acidified with 4Nhydrochloric acid (34 mL) to pH=1. The resulting mixture was extractedwith ethyl acetate (3×100 mL). The combined ethyl acetate extracts werewashed with brine (60 mL) and dried (MgSO₄). The solvent was removed invacuo to give 15.2 g (83%) of N-benzyloxycarbonyl-α-methylglutamic acidas an oil: ¹H NMR (CDCl₃) δ 8.73 (m, 5H), 5.77 (b, 1H), 5.09 (s, 2H),2.45-2.27 (m, 4H), 2.0 (s, 3H).

In a similar fashion from α-ethyl-D,L-glutamic acid andα-propyl-D,L-glutamic acid, there is obtainedN-benzyloxycarbonyl-α-ethylglutamic acid andN-benzyloxycarbonyl-α-propylglutamic acid, respectively.

Example 5 N-Benzyloxycarbonyl-α-methyl-glutamic Anhydride

A stirred mixture of N-benzyloxycarbonyl-α-methyl-glutamic acid (15 g,51 mmol) and acetic anhydride (65 mL) was heated at reflux undernitrogen for 30 min. The reaction mixture was cooled to room temperatureand then concentrated in vacuo to affordN-benzylcarbonyl-α-methylglutamic anhydride as an oil (15.7 g) which canbe used in next reaction without further purification: ¹H NMR (CDCl₃) δ7.44-7.26 (m, 5H), 5.32-5.30 (m, 2H), 5.11 (s, 1H), 2.69-2,61 (m, 2H),2.40-2.30 (m, 2H), 1.68 (s, 3H).

In a similar fashion from N-benzyloxycarbonyl-α-ethylglutamic acid andN-benzyloxycarbonyl-α-propylglutamic acid, there is obtainedN-benzylcarbonyl-α-ethylglutamic anhydride andN-benzylcarbonyl-α-propylglutamic anhydride, respectively.

Example 6 N-Benzyloxycarbonyl-α-methylisoglutamine

A stirred solution of N-benzylcarbonyl-α-methylglutamic anhydride (14.2g, 51.5 mmol) in methylene chloride (100 mL) was cooled in an ice bath.Gaseous ammonia was bubbled into the cooled solution for 2 hours. Thereaction mixture was stirred at room temperature for 17 hours and thenextracted with water (2×50 mL). The combined aqueous extracts werecooled in an ice bath and acidified with 4N hydrochloric acid (32 mL) topH 1. The resulting mixture was extracted with ethyl acetate (3×80 mL).The combined ethyl acetate extracts were washed with brine (60 mL) andthen dried (MgSO₄). The solvent was removed in vacuo to give 11.5 g ofN-benzyloxycarbonyl-α-amino-α-methylisoglutamine: ¹H NMR (CDCl₃/DMSO) δ7.35 (m, 5H), 7.01 (s, 1H), 6.87 (s, 1H), 6.29 (s, 1H), 5.04 (s, 2H),2.24-1.88 (m, 4H), 1.53 (s, 3H).

In a similar fashion from N-benzylcarbonyl-α-ethylglutamic anhydride andN-benzylcarbonyl-α-propylglutamic anhydride there is obtainedN-benzyloxycarbonyl-α-amino-α-ethylisoglutamine andN-benzyloxycarbonyl-α-amino-α-propylisoglutamine, respectively.

Example 7 N-Benzyloxycarbonyl-α-amino-α-methylglutarimide

A stirred mixture of N-benzyloxycarbonyl-α-methylisoglutamine (4.60 g,15.6 mmol), 1,1′-carbonyldiimidazole (2.80 g, 17.1 mmol), and4-dimethylaminopyridine (0.05 g) in tetrahydrofuran (50 mL) was heatedto reflux under nitrogen for 17 hours. The reaction mixture was thenconcentrated in vacuo to an oil. The oil was slurried in water (50 mL)for 1 hour. The resulting suspension was filtered and the solid washedwith water and air dried to afford 3.8 g of the crude product as a whitesolid. The crude product was purified by flash chromatography (methylenechloride:ethyl acetate 8:2) to afford 2.3 g (50%) ofN-benzyloxycarbonyl-α-amino-α-methylglutarimide as a white solid: mp150.5-152.5° C.; ¹H NMR (CDCl₃) δ 8.21 (s, 1H), 7.34 (s, 5H), 5.59 (s,1H), 5.08 (s, 2H), 2.74-2.57 (m, 3H), 2.28-2.25 (m, 1H), 1.54 (s, 3H);¹³C NMR (CDCl₃) δ 174.06, 171.56, 154.68, 135.88, 128.06, 127.69,127.65, 66.15, 54.79, 29.14, 28.70, 21.98; HPLC: Waters Nova-Pak C18column, 4 micron, 3.9×150 mm, 1 mL/min, 240 nm, 20/80 CH₃CN/0.1%H₃PO₄(aq), 7.56 min (100%); Anal. Calcd For C₁₄H₁₆N₂O₄; C, 60.86; H,5.84; N, 10.14. Found: C, 60.88; H, 5.72; N, 10.07.

In a similar fashion fromN-benzyloxycarbonyl-α-amino-α-ethylisoglutamine andN-benzyloxycarbonyl-α-amino-α-propylisoglutamine there is obtainedN-benzyloxycarbonyl-α-amino-α-ethylglutarimide andN-benzyloxycarbonyl-α-amino-o-propylglutarimide, respectively.

Example 8 α-Amino-α-methylglutarimide hydrochloride

N-Benzyloxycarbonyl-α-amino-α-methylglutarimide (2.3 g, 8.3 mmol) wasdissolved in ethanol (200 mL) with gentle heat and the resultingsolution allowed to cool to room temperature. To this solution was added4N hydrochloric acid (3 mL) followed by 10% Pd/C (0.4 g). The mixturewas hydrogenated in a Parr apparatus under 50 psi of hydrogen for 3hours. To the mixture was added water (50 mL) to dissolve the product.This mixture was filtered through a Celite pad which was washed withwater (50 mL). The filtrate was concentrated in vacuo to afford a solidresidue. The solid was slurried in ethanol (20 mL) for 30 min. Theslurry was filtered to afford 1.38 g (93%) ofα-amino-α-methylglutarimide hydrochloride as a white solid: ¹H NMR(DMSO-d₆) δ 11.25 (s, 1H), 8.92 (s, 3H), 2.84-2.51 (m, 2H), 2.35-2.09(m, 2H), 1.53 (s, 3H); HPLC, Waters Nova-Pak C₁₈ column, 4 micron, 1mL/min, 240 nm, 20/80 CH₃CN/0.1% H₃PO₄(aq), 1.03 min (94.6%).

In a similar fashion from N-benzyloxycarbonyl-α-amino-α-ethylglutarimideand N-benzyloxycarbonyl-α-amino-α-propylglutarimide there is obtainedα-amino-α-ethylglutarimide hydrochloride and α-amino-α-propylglutarimidehydrochloride, respectively.

Example 9 3-(3-Nitrophthalimido)-3-methylpiperidine-2,6-dione

A stirred mixture of α-amino-α-methylglutarimide hydrochloride (1.2 g,6.7 mmol), 3-nitrophthalic anhydride (1.3 g, 6.7 mmol), and sodiumacetate (0.6 g, 7.4 mmol) in acetic acid (30 mL) was heated to refluxunder nitrogen for 6 hours. The mixture then was cooled and concentratedin vacuo. The resulting solid was slurried in water (30 mL) andmethylene chloride (30 mL) for 30 min. The suspension was filtered, thesolid was washed with methylene chloride, and dried in vacuo (60° C., <1mm) to afford 1.44 g (68%) of3-(3-nitrophthalimido)-3-methylpiperidine-2,6-dione as a off-whitesolid: mp 265-266.5° C.; ¹H NMR (DMSO-d₆) δ 11.05 (s, 1H), 8.31 (dd,J=1.1 and 7.9 Hz, 1H), 8.16-8.03 (m, 2H), 2.67-2.49 (m, 3H), 2.08-2.02(m, 1H), 1.88 (s, 3H); ¹³C NMR (DMSO-d₆) δ 172.20, 171.71, 165.89,163.30, 144.19, 136.43, 133.04, 128.49, 126.77, 122.25, 59.22, 28.87,28.49, 21.04; HPLC, Water Nova-Pak/C₁₈ column, 4 micron, 1 mL/min, 240nm, 20/80 CH₃CN/0.1% H₃PO₄(aq), 7.38 min(98%). Anal. Calcd ForC₁₄H₁₁N₃O₆: C, 53.00; H, 3.49; N, 13.24. Found: C, 52.77; H, 3.29; N,13.00.

In a similar fashion from α-amino-α-ethylglutarimide hydrochloride andα-amino-α-propylglutarimide hydrochloride there is obtained3-(3-nitrophthalimido)-3-ethylpiperidine-2,6-dione and3-(3-nitrophthalimido)-3-propylpiperidine-2,6-dione, respectively.

Example 10 3-(3-Aminophthalimido)-3-methylpiperidine-2,6-dione

3-(3-Nitrophthalimido)-3-methylpiperidine-2,6-dione (0.5 g, 1.57 mmol)was dissolved in acetone (250 mL) with gentle heat and then cooled toroom temperature. To this solution was added 10% Pd/C (0.1 g) undernitrogen. The mixture was hydrogenated in a Parr apparatus at 50 psi ofhydrogen for 4 hours. The mixture then was filtered through Celite andthe pad washed with acetone (50 mL). The filtrate was concentrated invacuo to yield a yellow solid. The solid was slurried in ethyl acetate(10 mL) for 30 minutes. The slurry then was filtered and dried (60° C.,<1 mm) to afford 0.37 g (82%) of3-(3-aminophthalimido)-3-methylpiperidine-2,6-dione as a yellow solid:mp 268-269° C.; 1H NMR (DMSO-d₆) δ 10.98 (s, 1H), 7.44 (dd, J=7.1 and7.3 Hz, 1H), 6.99 (d, J=8.4 Hz, 1H), 6.94 (d, J=6.9 Hz, 1H), 6.52 (s,2H), 2.71-2.47 (m, 3H), 2.08-1.99 (m, 1H), 1.87 (s, 3H); ¹³C NMR(DMSO-d₆) δ 172.48, 172.18, 169.51, 168.06, 146.55, 135.38, 131.80,121.51, 110.56, 108.30, 58.29, 29.25, 28.63, 21.00; HPLC, WaterNova-Pak/C₁₈ column, 4 micron, 1 mL/min, 240 nm, 20/80 CH₃CN/0.1%H₃PO₄(aq), 5.62 min (99.18%). Anal. Calcd For C₁₄H₁₃N₃O₄: C, 58.53; H,4.56; N, 14.63. Found: C, 58.60; H, 4.41; N, 14.36.

In a similar fashion from3-(3-nitrophthalimido)-3-ethylpiperidine-2,6-dione and3-(3-nitrophthalimido)-3-propylpiperidine-2,6-dione there is obtained3-(3-aminophthalimido)-3-ethylpiperidine-2,6-dione and3-(3-aminophthalimido)-3-propylpiperidine-2,6-dione, respectively.

Example 11 Methyl 2-bromomethyl-3-nitrobenzoate

A stirred mixture of methyl 2-methyl-3-nitrobenzoate (17.6 g, 87.1 mmol)and N-bromosuccinimide (18.9 g, 105 mmol) in carbon tetrachloride (243mL) was heated under gentle reflux with a 100 W light bulb situated 2 cmaway shining on the reaction mixture overnight. After 18 hours, thereaction mixture was cooled to room temperature and filtered. Thefiltrate was washed with water (2×120 mL), brine (120 mL), and dried(MgSO₄). The solvent was removed in vacuo to give a yellow solid. Theproduct was purified by flash chromatography (hexane:ethyl acetate 8:2)to give 22 g (93%) of methyl 2-bromomethyl-3-nitrobenzoate as a yellowsolid: mp 69-72° C.; 1H NMR (CDCl₃) δ 8.13-8.09 (dd, J=1.36 and 7.86 Hz,1H), 7.98-7.93 (dd, J=1.32 and 8.13 Hz, 1H), 7.57-7.51 (t, J=7.97 Hz,1H), 5.16 (s, 2H), 4.0 (s, 3H); ¹³C NMR (CDCl₃) δ 65.84, 150.56, 134.68,132.64, 132.36, 129.09, 53.05, 22.70; HPLC: Waters Nova-Pak C₁₈ column,4 micron, 1 mL/min, 240 nm, 40/60 CH₃CN/0.1% H₃PO₄(aq), 8.2 min 99%.Anal. Calcd for C₉H₈NO₄Br: C, 39.44; H, 2.94; N, 5.11, Br, 29.15. Found:C, 39.51; H, 2.79; N, 5.02; Br, 29.32.

Example 12 3-(1-Oxo-4-nitroisoindolin-1-yl)-3-methylpiperidine-2,6-dione

To a stirred mixture of α-amino-α-methylglutarimide hydrochloride (2.5g, 14.0 mmol) and methyl 2-bromomethyl-3-nitrobenzoate (3.87 g, 14.0mmol in dimethylformamide (40 mL) was added triethylamine (3.14 g, 30.8mmol). The resulting mixture was heated to reflux under nitrogen for 6hours. The mixture was cooled and then concentrated in vacuo. Theresulting solid was slurried in water (50 mL) and CH₂Cl₂ for 30 min. Theslurry was filtered, the solid washed with methylene chloride, and driedin vacuo (60° C., <1 mm) to afford 2.68 g (63%) of3-(1-oxo-4-nitroisoindolin-1-yl)-3-methylpiperidine-2,6-dione as aoff-white solid: mp 233-235° C.; ¹H NMR (DMSO-d₆) δ 10.95 (s, 1H),8.49-8.46 (d, J=8.15 Hz, 1H), 8.13-8.09 (d, J=7.43 Hz, 1H), 7.86-7.79(t, J=7.83 Hz, 1H), 5.22-5.0 (dd, J=19.35 and 34.6 Hz, 2H), 2.77-2.49(m, 3H), 2.0-1.94 (m, 1H), 1.74 (S, 3H); ¹³C NMR (DMSO-d₆) δ 173.07,172.27, 164.95, 143.15, 137.36, 135.19, 130.11, 129.32, 126.93, 57.57,48.69, 28.9, 27.66, 20.6; HPLC, Waters Nova-Pak C₁₈ column, 4 micron, 1mL/min, 240 nm, 20/80 CH₃CN/0.1% H₃PO₄(aq), 4.54 min 99.6%. Anal. Calcdfor C₁₄H₁₁N₃O₅: C, 55.45; H, 4.32; N, 13.86. Found: C, 52.16; H, 4.59;N, 12.47.

By substituting equivalent amounts of α-amino-α-ethylglutarimidehydrochloride and α-amino-α-propylglutarimide hydrochloride forα-amino-α-methylglutarimide hydrochloride, there is obtainedrespectively3-(1-oxo-4-nitroisoindolin-1-yl)-3-ethylpiperidine-2,6-dione and3-(1-oxo-4-nitroisoindolin-1-yl)-3-propylpiperidine-2,6-dione.

Example 13 3-(1-Oxo-4-aminoisoindolin-1-yl)-3-methylpiperidine-2,6-dione

3-(1-Oxo-4-nitroisoindolin-1-yl)-3-methylpiperidine-2,6-dione (1.0 g,3.3 mmol) was dissolved in methanol (500 mL) with gentle heat andallowed to cool to room temperature. To this solution was added 10% Pd/C(0.3 g) under nitrogen. The mixture was hydrogenated in a Parr apparatusat 50 psi of hydrogen for 4 hours. The mixture was filtered throughCelite and the Celite washed with methanol (50 mL). The filtrate wasconcentrated in vacuo to an off white solid. The solid was slurried inmethylene chloride (20 mL) for 30 min. The slurry was then filtered andthe solid dried (60° C., <1 mm) to afford 0.54 g (60%) of3-(1-oxo-4-aminoisoindolin-1-yl)-3-methylpiperidine-2,6-dione as a whitesolid: mp 268-270° C.; ¹H NMR (DMSO-d₆) δ 10.85 (s, 1H), 7.19-7.13 (t,J=7.63 Hz, 1H), 6.83-6.76 (m, 2H), 5.44 (s, 2H), 4.41 (s, 2H), 2.71-2.49(m, 3H), 1.9-1.8 (m, 1H), 1.67 (s, 3H); ¹³C NMR (DMSO-d₆) δ 173.7,172.49, 168.0, 143.5, 132.88, 128.78, 125.62, 116.12, 109.92, 56.98,46.22, 29.04, 27.77, 20.82; HPLC, Waters Nova-Pak/C₁₈ column, 4 micron,1 mL/min, 240 nm, 20/80 CH₃CN/0.1% H₃PO₄(aq), 1.5 min (99.6%); Anal.Calcd for C₁₄H₁₅N₃O₃: C, 61.53; H, 5.53; N, 15.38. Found: C, 58.99; H,5.48; N, 14.29.

From 3-(1-oxo-4-nitroisoindolin-1-yl)-3-ethylpiperidine-2,6-dione and3-(1-oxo-4-nitroisoindolin-1-yl)-3-propylpiperidine-2,6-dione there issimilarly obtained3-(1-oxo-4-aminoisoindolin-1-yl)-3-ethylpiperidine-2,6-dione and3-(1-oxo-4-aminoisoindolin-1-yl)-3-propylpiperidine-2,6-dione,respectively.

Example 14 S-4-Amino-2-(2,6-dioxopiperid-3-yl)isoindoline-1,3-dione A.4-Nitro-N-ethoxycarbonylphthalimide

Ethyl chloroformate (1.89 g, 19.7 mmol) was added dropwise over 10 minto a stirred solution of 3-nitrophthalimide (3.0 g, 15.6 mmol) andtriethylamine (1.78 g, 17.6 mmol) in dimethylformamide (20 mL) at 0-5°C. under nitrogen. The reaction mixture was allowed to warm to roomtemperature and stirred for 4 hours. The mixture was then slowly addedto an agitated mixture of ice and water (60 mL). The resulting slurrywas filtered and the solid was crystallized from chloroform (15 mL) andpet ether (15 mL) to afford 3.1 g (75%) of the product as an off-whitesolid: mp 100-100.5° C.; ¹H NMR (CDCl₃) δ 8.25 (d, J=7.5 Hz, 1H), 8.20(d, J=8.0 Hz, 1H), 8.03 (t, J=7.9 Hz, 1H), 4.49 (q, J=7.1 Hz, 2H), 1.44(t, J=7.2 Hz, 3H); ¹³C NMR (CDCl₃) δ 161.45, 158.40, 147.52, 145.65,136.60, 132.93, 129.65, 128.01, 122.54, 64.64, 13.92; HPLC, WatersNova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 30/70 CH₃CN/0.1%H₃PO₄(aq), 5.17 min(98.11%); Anal. Calcd for C₁₁H₈N₂O₆: C, 50.00; H,3.05; N, 10.60. Found: C, 50.13; H, 2.96; N, 10.54.

B. t-Butyl N-(4-nitrophthaloyl)-L-glutamine

A stirred mixture of 4-nitro-N-ethoxycarbonylphthalimide (1.0 g, 3.8mmol), L-glutamine t-butyl ester hydrochloride (0.90 g, 3.8 mmol) andtriethylamine (0.54 g, 5.3 mmol) in tetrahydrofuran (30 mL) was heated,to reflux for 24 hours. The tetrahydrofuran was removed in vacuo and theresidue was dissolved in methylene chloride (50 mL). The methylenechloride solution was washed with water (2×15 mL), brine (15 mL) andthen dried (sodium sulfate). The solvent was removed in vacuo and theresidue was purified by flash chromatograph (7:3 methylenechloride:ethyl acetate) to give 0.9 g (63%) of a glassy material: ¹H NMR(CDCl₃) δ 8.15 (d, J=7.9 Hz, 2H), 7.94 (t, J=7.8 Hz, 1H), 5.57 (b, 2H),4.84 (dd, J=5.1 and 9.7 Hz, 1H), 2.53-2.30 (m, 4H), 1.43 (s, 9H); HPLC,Wasters Nova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 30/70CH₃CN/0.1% H₃PO₄(aq), 6.48 min(99.68%); Chiral Analysis, Daicel ChiralPak AD, 0.4×25 Cm, 1 mL/min, 240 nm, 5.32 min(99.39%); Anal. Calcd forC₁₇H₁₉N₃O₇: C, 54.11; H, 5.08; N, 11.14. Found: C, 54.21; H, 5.08; N,10.85.

C. N-(4-Nitrophthaloyl)-L-glutamine

Hydrogen chloride gas was bubbled into a stirred 5° C. solution oft-butyl N-(4-nitrophthaloyl)-L-glutamine (5.7 g, 15.1 mmol) in methylenechloride (100 mL) for 25 min. The mixture was then stirred at roomtemperature for 16 hours. Ether (50 mL) was added and the resultingmixture was stirred for 30 min. The resulting slurry was filtered toyield 4.5 g of crude product as a solid, which was used directly in thenext reaction: ¹H NMR (DMSO-d₆) δ 8.36 (dd, J=0.8 and 8.0 Hz, 1H), 8.24(dd, J=0.8 and 7.5 Hz, 1H), 8.11 (t, J=7.9 Hz, 1H), 7.19 (b, 1H), 6.72(b, 1H), 4.80 (dd, J=3.5 and 8.8 Hz, 1H), 2.30-2.10 (m, 4H).

D. (S)-2-(2,6-dioxo(3-piperidyl))-4-nitroisoindoline-1,3-dione

A stirred suspension of N-(4-nitrophthaloyl)-L-glutamine (4.3 g, 13.4mmol) in anhydrous methylene chloride (170 mL) was cooled to −40° C.(IPA/dry ice bath). Thionyl chloride (1.03 mL, 14.5 mmol) was addeddropwise to the mixture followed by pyridine (1.17 mL, 14.5 mmol). After30 minutes, triethylamine (2.66 mL, 14.8 mmol) was added and the mixturewas stirred at −30 to −40° C. for 3 hours. The mixture was allowed towarm to room temperature, filtered and washed with methylene chloride toafford 2.3 g (57%) of the crude product. Recrystallization from acetone(300 mL) afforded 2 g of the product as a white solid: mp 259.0-284.0°C. (dec.); ¹H NMR (DMSO-d₆) δ 11.19 (s, 1H), 8.34 (d, J=7.8 Hz, 1H),8.23 (d, J=7.1 Hz, 1H), 8.12 (t, J=7.8 Hz, 1H), 5.25-5.17 (dd, J=5.2 and12.7 Hz, 1H), 2.97-2.82 (m, 1H), 2.64-2.44 (m, 2H), 2.08-2.05 (m, 1H);¹³C NMR (DMSO-d₆) δ 172.67, 169.46, 165.15, 162.50, 144.42, 136.78,132.99, 128.84, 127.27, 122.53, 49.41, 30.84, 21.71; HPLC, WatersNova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 10/90 CH₃CN/0.1%H₃PO₄(aq) 4.27 min(99.63%); Anal. Calcd for C₁₃H₉N₃O₆: C, 51.49; H,2.99; N, 13.86. Found: C, 51.67; H, 2.93; N, 13.57.

E. S-4-Amino-2-(2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A mixture of (S)-3-(4′-nitrophthalimido)-piperidine-2,6-dione (0.76 g,2.5 mmol) and 10% Pd/C (0.3 g) in acetone (200 mL) was hydrogenated in aParr-Shaker apparatus at 50 psi of hydrogen for 24 hours. The mixturewas filtered through celite and the filtrate was concentrated in vacuo.The solid residue was slurried in hot ethyl acetate for 30 min andfiltered to yield 0.47 g (69%) of the product as a yellow solid mp309-310° C.; ¹H NMR (DMSO-d₆) δ 11.10 (s, 1H), 7.47 (dd, J=7.2 and 8.3Hz, 1H), 7.04-6.99 (dd, J=6.9 and 8.3 Hz, 2H), 6.53 (s, 2H), 5.09-5.02(dd, J=5.3 and 12.4 Hz, 1H), 2.96-2.82 (m, 1H), 2.62-2.46 (m, 2H),2.09-1.99 (m, 1H); ¹³C NMR (DMSO-d₆) δ 172.80, 170.10, 168.57, 167.36,146.71, 135.44, 131.98, 121.69, 110.98, 108.54, 48.48, 30.97, 22.15;HPLC, Waters Nova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 15/85CH₃CN/0.1% H₃PO₄(aq) 4.99 min(98.77%); Chiral analysis, Daicel Chiral.Pak AD, 0.46×25 cm, 1 mL/min, 240 nm, 30/70 Hexane/IPA 9.55 min (1.32%),12.55 min (97.66%); Anal. Calcd for C₁₃H₁₁N₃O₄: C, 57.14; H, 4.06; N,15.38. Found: C, 57.15; H, 4.15; N, 14.99.

Example 15 R-4-Amino-2-(2,6-dioxopiperid-3-yl))isoindoline-1,3-dione A.t-Butyl N-(4-nitrophthaloyl)-D-glutamine

A stirred mixture of 4-nitro-N-ethoxycarbonyl-phthalimide (5.9 g, 22.3mmol), D-glutamine t-butyl ester (4.5 g, 22.3 mmol) and triethylamine(0.9 g, 8.9 mmol) in tetrahydrofuran (100 mL) was refluxed for 24 hours.The mixture was diluted with methylene chloride (100 mL) and washed withwater (2×50 mL), brine (50 mL) and then dried. The solvent was removedin vacuo and the residue was purified by flash chromatography (2% CH₃OHin methylene chloride) to afford 6.26 g (75%) of the product as a glassymaterial: ¹H NMR (CDCl₃) δ 8.12 (d, J=7.5 Hz, 2H), 7.94 (dd, J=7.9 and9.1 Hz, 1H), 5.50 (b, 1H), 5.41 (b, 1H), 4.85 (dd, J=5.1 and 9.8 Hz,1H), 2.61-2.50 (m, 2H), 2.35-2.27 (m, 2H), 1.44 (s, 9H); ¹³C NMR (CDCl₃)δ 173.77, 167.06, 165.25, 162.51, 145.07, 135.56, 133.78, 128.72,127.27, 123.45, 83.23, 53.18, 32.27, 27.79, 24.42; HPLC, WatersNova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 25/75 CH₃CN/0.1%H₃PO₄(aq) 4.32 min(99.74%); Chiral analysis, Daicel Chiral Pak AD,0.46×25 cm, 1 mL/min, 240 nm, 55/45 Hexane/IPA 5.88 min(99.68%); Anal.Calcd for C₁₇H₁₉N₃O₇: C, 54.11; H, 5.08; N, 11.14. Found: C, 54.25; H,5.12; N, 10.85.

B. N-(4-Nitrophthaloyl)-D-glutamine

Hydrogen chloride gas was bubbled into a stirred 5° C. solution oft-butyl N-(4-nitrophthaloyl)-D-glutamine (5.9 g, 15.6 mmol) in methylenechloride (100 mL) for 1 hour then stirred at room temperature foranother hour. Ether (100 mL) was added and stirred for another 30minutes. The mixture was filtered, the solid was washed with ether (60mL) and dried (40° C., <1 mm Hg) to afford 4.7 g (94%) of the product:¹H NMR (DMSO-d₆) δ 8.33 (d, J=7.8 Hz, 1H), 8.22 (d, J=7.2 Hz, 1H), 8.11(t, J=7.8 Hz, 1H), 7.19 (b, 1H), 6.72 (b, 1H), 4.81 (dd, J=4.6 and 9.7Hz, 1H), 2.39-2.12 (m, 4H); ¹³C NMR (DMSO-d₆) δ 173.21, 169.99, 165.41,162.73, 144.45, 136.68, 132.98, 128.80, 127.23, 122.52, 51.87, 31.31,23.87.

C. (R)-2-(2,6-dioxo(3-piperidyl))-4-nitroisoindoline-1,3-dione

A stirred suspension of N-(4′-nitrophthaloyl)-D-glutamine (4.3 g, 13.4mmol) in anhydrous methylene chloride (170 mL) was cooled to −40° C.with isopropanol/dry ice bath. Thionyl chloride (1.7 g, 14.5 mmol) wasadded dropwise followed by pyridine (1.2 g, 14.5 mmol). After 30 min,triethylamine (1.5 g, 14.8 mmol) was added and the mixture was stirredat −30 to −40° C. for 3 hours. The mixture was filtered, the solidwashed with methylene chloride (50 mL) and dried (60° C., <1 mm Hg) togive 2.93 g of the product. Another 0.6 g of the product was obtainedfrom the methylene chloride filtrate. Both fractions were combined (3.53g) and recrystallized from acetone (450 mL) to afford 2.89 g (71%) ofthe product as a white solid: mp 256.5-257.5° C.; ¹H NMR (DMSO-d₆) δ11.18 (s, 1H), 8.34 (dd, J=0.8 and 7.9 Hz, 1H), 8.23 (dd, J=0.8 and 7.5Hz, 1H), 8.12 (t, J=7.8 Hz, 1H), 5.22 (dd, J=5.3 and 12.8 Hz, 1H),2.97-2.82 (m, 1H), 2.64-2.47 (m, 2H), 2.13-2.04 (m, 1H); ¹³C NMR(DMSO-d₆) δ 172.66, 169.44, 165.14, 162.48, 144.41, 136.76, 132.98,128.83, 127.25, 122.52, 49.41, 30.83; 21.70; HPLC, Waters Nova-Pak/C18,3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 10/90 CH₃CN/0.1% H₃PO₄(aq) 3.35min(100%); Anal. Calcd for C₁₃H₉N₃O₆: C, 51.49; H, 2.99; N, 13.86. FoundC, 51.55; H, 2.82; N, 13.48.

D. (R)-4-Amino-2-(2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A mixture of R-3-(4′-nitrophthalimido)-piperidine-2,6-dione (1.0 g, 3.3mmol) and 10% Pd/C (0.2 g) in acetone (250 mL) was hydrogenated in aParr-Shaker apparatus at 50 psi of hydrogen for 4 hours. The mixture wasfiltered through celite and the filtrate was concentrated in vacuo. Theresulting yellow solid was slurried in hot ethyl acetate (20 mL) for 30min to give after filtration and drying 0.53 g (59%) of the product as ayellow solid: mp 307.5-309.5° C.; 1H NMR (DMSO-d₆) δ 11.06 (s, 1H), 7.47(dd, J=7.0 and 8.4 Hz, 1H), 7.02 (dd, J=4.6 and 8.4 Hz, 2H), 6.53 (s,2H), 5.07 (dd, J=5.4 and 12.5 Hz, 1H), 2.95-2.84 (m, 1H), 2.62-2.46 (m,2H), 2.09-1.99 (m, 1H); ¹³C NMR (DMSO-d₆) δ 172.78, 170.08, 168.56,167.35, 146.70, 135.43, 131.98, 121.68, 110.95, 108.5.3, 48.47, 30.96,22.14; HPLC, Waters Nove-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240nm, 10/90 CH₃CN/0.1% H₃PO₄(aq) 3.67 min(99.68%); Chiral analysis, DaicelChiral Pak AD, 0.46×25 cm, 1 mL/min, 240 nm, 30/70 Hexane/IPA 7.88 min(97.48%); Anal. Calcd for C₁₃H₁₁N₃O₄: C, 57.14; H, 4.06; N, 15.38.Found: C, 57.34; H, 3.91; N, 15.14.

Example 16 3-(4-Amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione A.Methyl 2-bromomethyl-3-nitrobenzoate

A stirred mixture of methyl 2-methyl-3-nitrobenzoate (14.0 g, 71.7 mmol)and N-bromosuccinimide (15.3 g, 86.1 mmol) in carbon tetrachloride (200mL) was heated under gentle reflux for 15 hours while a 100 W bulbsituated 2 cm away was shining on the flask. The mixture was filteredand the solid was washed with methylene chloride (50 mL). The filtratewas washed with water (2×100 mL), brine (100 mL) and dried. The solventwas removed in vacuo and the residue was purified by flashchromatography (hexane/ethyl acetate, 8/2) to afford 19 g (96%) of theproduct as a yellow solid: mp 70.0-71.5° C.; ¹H NMR (CDCl₃) δ 8.12-8.09(dd, J=1.3 and 7.8 Hz, 1H), 7.97-7.94 (dd, J=1.3 and 8.2 Hz, 1H), 7.54(t, J=8.0 Hz, 1H), 5.15 (s, 2H), 4.00 (s, 3H); ¹³C NMR (CDCl₃) δ 165.85,150.58, 134.68, 132.38, 129.08, 127.80, 53.06, 22.69; HPLC, WaterNove-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 40/60 CH₃CN/0.1%H₃PO₄(aq) 7.27 min(98.92%); Anal. Calcd for C₉H₈NO₄Br: C, 39.44; H,2.94; N, 5.11; Br, 29.15. Found: C, 39.46; H, 3.00; N, 5.00; Br, 29.11.

B. t-Butyl N-(1-oxo-4-nitroisoindolin-2-yl)-L-glutamine

Triethylamine (2.9 g, 28.6 mmol) was added dropwise to a stirred mixtureof methyl 2-bromomethyl-3-nitrobenzoate (3.5 g, 13.0 mmol) andL-glutamine t-butyl ester hydrochloride (3.1 g, 13.0 mmol) intetrahydrofuran (90 mL). The mixture was heated to reflux for 24 hours.To the cooled mixture was added methylene chloride (150 mL) and themixture was washed with water (2×40 mL), brine (40 mL) and dried. Thesolvent was removed in vacuo and the residue was purified by flashchromatography (3% CH₃OH in methylene chloride) to afford 2.84 g (60%)of crude product which was used directly in the next reaction: ¹H NMR(CDCl₃) δ 8.40 (d, J=8.1 Hz, 1H), 8.15 (d, J=7.5 Hz, 1H), 7.71 (t; J=7.8Hz, 1H), 5.83 (s, 1H), 5.61 (s, 1H), 5.12 (d, J=19.4 Hz, 1H), 5.04-4.98(m, 1H), 4.92 (d, J=19.4 Hz, 1H), 2.49-2.22 (m, 4H), 1.46 (s, 9H); HPLC,Waters Nova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 25/75CH₃CN/0.1% H₃PO₄(aq) 6.75 min(99.94%).

C. N-(1-Oxo-4-nitroisoindolin-2-yl)-L-glutamine

Hydrogen chloride gas was bubbled into a stirred 5° C. solution oft-butyl N-(1-oxo-4-nitro-isoindolin-2-yl)-L-glutamine (3.6 g, 9.9 mmol)in methylene chloride (60 mL) for 1 hour. The mixture was then stirredat room temperature for another hour. Ether (40 mL) was added and theresulting mixture was stirred for 30 minutes. The slurry was filtered,washed with ether and dried to afford 3.3 g of the product: ¹H NMR(DMSO-d₆) δ 8.45 (d, J=8.1 Hz, 1H), 8.15 (d, J=7.5 Hz, 1H), 7.83 (t,J=7.9 Hz, 1H), 7.24 (s, 1H), 6.76 (s, 1H), 4.93 (s, 2H), 4.84-4.78 (dd,J=4.8amd 10.4 Hz, 1H), 2.34-2.10 (m, 4H); ¹³C NMR (DMSO-d₆) δ 173.03,171.88, 165.96, 143.35, 137.49, 134.77, 130.10, 129.61, 126.95, 53.65,48.13, 31.50, 24.69; Anal. Calcd for C₁₃H₁₃N₃O₆: C, 50.82; H, 4.26; N,13.68. Found: C, 50.53; H, 4.37; N, 13.22.

D. (S)-3-(1-Oxo-4-nitroisoindolin-2-yl)piperidine-2,6-dione

A stirred suspension mixture ofN-(1-oxo-4-nitroisoindolin-2-yl)-L-glutamine (3.2 g, 10.5 mmol) inanhydrous methylene chloride (150 mL) was cooled to −40° C. withisopropanol/dry ice bath. Thionyl chloride (0.82 mL, 11.3 mmol) wasadded dropwise to the cooled mixture followed by pyridine (0.9 g, 11.3mmol). After 30 min, triethylamine (1.2 g, 11.5 mmol) was added and themixture was stirred at −30 to −40° C. for 3 hours. The mixture waspoured into ice water (200 mL) and the aqueous layer was extracted withmethylene chloride (40 mL). The methylene chloride solution was washedwith water (2×60 mL), brine (60 mL) and dried. The solvent was removedin vacuo and the solid residue was slurried with ethyl acetate (20 mL)to give 2.2 g (75%) of the product as a white solid: mp 285° C.; ¹H NMR(DMSO-d₆) δ 11.04 (s, 1H), 8.49-8.45 (dd, J=0.8 and 8.2 Hz, 1H),8.21-8.17 (dd, J=7.3 Hz, 1H), 7.84 (t, J=7.6 Hz, 1H), 5.23-5.15 (dd,J=4.9 and 13.0 Hz, 1H), 4.96 (dd, J=19.3 and 32.4 Hz, 2H), 3.00-2.85 (m,1H), 2.64-2.49 (m, 2H), 2.08-1.98 (m, 1H); ¹³C NMR (DMSO-d₆) δ 172.79,170.69, 165.93, 143.33, 137.40, 134.68, 130.15, 129.60, 127.02, 51.82,48.43, 31.16, 22.23; HPLC, Waters Nove-Pak/C18, 3.9×150 mm, 4 micron, 1mL/min, 240 nm, 20/80 CH₃CN/0.1% H₃PO₄(aq) 3.67 min(100%); Anal. Calcdfor C₁₃H₁₁N₃O₅: C, 53.98; H, 3.83; N, 14.53. Found: C, 53.92; H, 3.70;N, 14.10.

E. (S)-3-(1-Oxo-4-aminoisoindolin-2-yl)piperidine-2,6dione

A mixture of (S)-3-(1-oxo-4-nitroisoindolin-2-yl)piperidine-2,6-dione(1.0 g, 3.5 mmol) and 10% Pd/c (0.3 g) in methanol (600 mL) washydrogenated in a Parr-Shaker apparatus at 50 psi of hydrogen for 5hours. The mixture was filtered through Celite and the filtrate wasconcentrated in vacuo. The solid was slurried in hot ethyl acetate for30 min, filtered and dried to afford 0.46 g (51%) of the product as awhite solid: mp 235.5-239° C.; ¹H NMR (DMSO-d₆) δ 11.01 (s, 1H), 7.19(t, J=7.6 Hz, 1H), 6.90 (d, J=7.3 Hz, 1H), 6.78 (d, J=7.8 Hz, 1H), 5.42(s, 2H), 5.12 (dd, J=5.1 and 13.1 Hz, 1H), 4.17 (dd, J=17.0 and 28.8 Hz,2H), 2.92-2.85 (m, 1H), 2.64-2.49 (m, 1H), 2.34-2.27 (m, 1H), 2.06-1.99(m, 1H); ¹³C NMR (DMSO-d₆) δ 172.85, 171.19, 168.84, 143.58, 132.22,128.79, 125.56, 116.37, 110.39, 51.48, 45.49, 31.20, 22.74; HPLC, WatersNova-Pak/C18, 3.9×150 mm, 4 micron, 1 mL/min, 240 nm, 10/90 CH₃CN/0.1%H₃PO₄(aq) 0.96 min(100%); Chiral analysis, Daicel Chiral Pak AD, 40/60Hexane/IPA, 6.60 min(99.42%); Anal. Calcd for C₁₃H₁₃N₃O₃: C, 60.23; H,5.05: N, 16.21. Found: C, 59.96; H, 4.98; N, 15.84.

Example 17 3-(4-Amino-1-oxoisoindolin-2yl)-3-methylpiperidine-2,6-dioneA. N-Benzyloxycarbonyl-3-amino-3-methylpiperidine-2,6-dione

A stirred mixture of N-benzyloxycarbonyl-α-methyl-isoglutamine (11.3 g,38.5 mmol), 1,1′-carbonyldiimidazole (6.84 g, 42.2 mmol) and4-dimethylaminopyridine (0.05 g) in tetrahydrofuran (125 mL) was heatedto reflux under nitrogen for 19 hours. The reaction mixture wasconcentrated in vacuo to an oil. The oil was slurried in water (50 mL)for 1 hour then filtered, washed with water, air dried to afford 7.15 gof white solid. The crude product was purified by flash chromatography(2:8 ethyl acetate:methylene chloride) to afford 6.7 g (63%) of theproduct as a white solid: mp 151-152° C.; 1H NMR (CDCl₃) δ 8.24 (s, 1H),7.35 (s, 5H), 5.6 (s, 1H), 5.09 (s, 2H), 2.82-2.53 (m, 3H), 2.33-2.26(m, 1H), 1.56 (s, 3H); ¹³C NMR (CDCl₃) δ 174.4, 172.4, 154.8, 136.9,128.3, 127.8, 127.7, 65.3, 54.6, 29.2, 29.0, 22.18; HPLC: WatersNova-Pak/C₁₈ column, 4 micron, 3.9×150 mm, 1 ml/min, 240 nm, 20/80CH₃CN/H₃PO₄(aq), 6.6 min, 100%). Anal. Calcd for C₁₄H₁₆N₂O₄. Theory: C,60.86; H, 5.84; N, 10.14. Found: C, 60.94; H, 5.76; N, 10.10.

B. 3-Amino-3-methylpiperidine-2,6-dione

N-benzyloxycarbonyl-3-amino-3-methylpiperidine-2,6-dione (3.0 g, 10.9mmol) was dissolved in ethanol (270 mL) with gentle heat and then cooledto room temperature. To this solution was added 4 N HCl (7 mL) followedby 10% Pd/C (0.52 g). The mixture was hydrogenated under 50 psi ofhydrogen for 3 hours. To the mixture was then added water (65 mL) todissolve the product. The mixture was filtered through a celite pad andthe celite pad washed with water (100 mL). The filtrate was concentratedin vacuo to a solid residue. This solid was slurried in ethanol (50 mL)for 30 min. The slurry was filtered to afford 3.65 g (94%) of theproduct as a white solid: ¹H NMR (DMSO-d₆) δ 11.25 (s, 1H), 8.9 (s, 3H),2.87-2.57 (m, 2H), 2.35-2.08 (m, 2H), 1.54 (s, 3H); HPLC (WatersNova-Pak/C₁₈ column, 4 micron, 1 ml/min, 240 nm, 15/85 CH₃CN/H₃PO₄(aq),1.07 min, 100%).

C. 3-Methyl-3-(4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione

To a stirred mixture of α-amino-α-methyl-glutarimide hydrochloride (2.5g, 14.0 mmol) and methyl 2-bromomethyl-3-nitro benzoate (3.87 g, 14 mmolin dimethylformamide (40 mL) was added triethylamine (3.14 g, 30.8 mmol)under nitrogen. The mixture was heated to reflux for 6 hours. Themixture was cooled and then concentrated in vacuo. The solid residue wasslurried in water (50 mL) and methylene chloride for 30 min. The slurrywas filtered and the solid washed with methylene chloride and dried (60°C., <1 mm). Recrystallization from methanol (80 mL) yielded 0.63 g (15%)of the product as an off white solid: mp 195-197° C.; 1H NMR (DMSO-d₆) δ10.95 (s, 1H), 8.49-8.46 (d, J=8.2 Hz, 1H), 8.13-8.09 (d, J=7.4 Hz, 1H),7.86-7.79 (t, J=7.8 Hz, 1H), 5.22-5.0 (dd, J=19.4 and 34.6 Hz, 2H),2.77-2.49 (m, 3H), 2.0-1.94 (m, 1H), 1.74 (S, 3H); ¹³C NMR (DMSO-d₆) δ173.1, 172.3, 165.0, 143.2, 137.4, 135.2, 130.1, 129.3, 126.9, 57.6,48.7, 28.9, 27.7, 20.6; HPLC (Waters Nova-Pak/C₁₈ column, 4 micron, 1ml/min, 240 nm, 20/80 CH₃CN/H₃PO_(4(aq)), 4.54 min, 99.6%); Anal Calcd.For C₁₄H₁₃N₃O₅; C, 55.45; H, 4.32; N, 13.86. Found: C, 55.30; H, 4.48;N, 13.54.

D. 3-Methyl-3-(4-amino-1-oxoisoindolin-2yl)piperidine-2,6-dione

3-Methyl-3-(4-nitro-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1.0 g,3.3 mmol) was dissolved in methanol (500 mL) with gentle heat and thencooled to room temperature. To this solution was added 10% Pd/C (0.3 g)under nitrogen. The mixture was hydrogenated in a Parr-Shaker apparatusat 50 psi of hydrogen for 4 hours. The mixture was filtered throughcelite pad and the celite pad washed with methanol (50 mL). The filtratewas concentrated in vacuo to a off white solid. The solid was slurriedin methylene chloride (20 mL) for 30 min. The slurry was filtered andthe solid dried (60° C., <1 mm). The solid was to recrystallized frommethanol (3 times, 100 mL/time) to yield 0.12 g (13.3%) of the productas a white solid: mp 289-292° C.; ¹H NMR (DMSO-d₆) δ 10.85 (s, 1H),7.19-7.13 (t, J=7.6 Hz, 1H), 6.83-6.76 (m, 2H), 5.44 (s, 2H), 4.41 (s,2H), 2.71-2.49 (m, 3H), 1.9-1.8 (m, 1H), 1.67 (s, 3H); ¹³C NMR (DMSO-d₆)δ 173.7, 172.5, 168.0, 143.5, 132.9, 128.8, 125.6, 116.1, 109.9, 57.0,46.2, 29.0, 27.8, 20.8; HPLC (Waters Nova-Pak/C₁₈ column, 4 micron, 1ml/min, 240 nm, 20/80 CH₃CN/H₃PO₄(aq), 1.5 min, 99.6%); Anal. Calcd. ForC₁₄H₁₅N₃O₃; C, 61.53; H, 5.53; N, 15.38. Found: C, 61.22; H, 5.63; N,15.25.

Example 18

Tablets, each containing 50 mg of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aninoisoindoline, can beprepared in the following manner:

Constituents (for 1000 tablets) 1,3-dioxo-2-(2,6-dioxo- 50.0 g piperidin-3-yl)-5-amino- isoindoline lactose 50.7 g  wheat starch 7.5 gpolyethylene glycol 6000 5.0 g talc 5.0 g magnesium stearate 1.8 gdemineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35° C., forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

Example 19

Tablets, each containing 100 mg of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline, can beprepared in the following manner:

Constituents (for 1000 tablets) 1,3-dioxo-2-(2,6-dioxo- 100.0 gpiperidin-3-yl)-5-amino- isoindoline lactose 100.0 g wheat starch  47.0g magnesium stearate  3.0 g

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, lactose, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to 100 mL of boilingwater. The resulting paste is added to the pulverulent substances andthe mixture is granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 6 mm diameterwhich are concave on both sides.

Example 20

Tablets for chewing, each containing 75 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline, can be prepared inthe following manner:

Composition (for 1000 tablets) 1-oxo-2-(2,6-dioxo- 75.0 gpiperidin-3-yl)-4-amino- isoindoline mannitol 230.0 g  lactose 150.0 g talc 21.0 g glycine 12.5 g stearic acid 10.0 g saccharin  1.5 g 5%gelatin solution q.s.

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol and the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50° C. and again forced through a sieve of 1.7 mm mesh width.1-Oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline, the glycine andthe saccharin are carefully mixed, the mannitol, the lactose granulate,the stearic acid and the talc are added and the whole is mixedthoroughly and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking groove onthe upper side.

Example 21

Tablets, each containing 10 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline, can be prepared inthe following manner:

Composition (for 1000 tablets) 1-oxo-2-(2,6-dioxo- 10.0 gpiperidin-3-yl)-5-amino- isoindoline lactose 328.5 g  corn starch 17.5 gpolyethylene glycol 6000  5.0 g talc 25.0 g magnesium stearate  4.0 gdemineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. Then the active imide ingredient, lactose, talc, magnesiumstearate and half of the starch are intimately mixed. The other half ofthe starch is suspended in 65 mL of water and this suspension is addedto a boiling solution of the polyethylene glycol in 260 mL of water. Theresulting paste is added to the pulverulent substances, and the whole ismixed and granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

Example 22

Gelatin dry-filled capsules each containing 100 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline, can be prepared inthe following manner:

Composition (for 1000 capsules) 1-oxo-2-(2,6-dioxo- 100.0 g piperidin-3-yl)-6-amino- isoindoline microcrystalline cellulose 30.0 g sodium lauryl sulfate 2.0 g magnesium stearate 8.0 g

The sodium lauryl sulfate is sieved into the1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline through a sieve of0.2 mm mesh width and the two components are intimately mixed for 10minutes. The microcrystalline cellulose is then added through a sieve of0.9 mm mesh width and the whole is again intimately mixed for 10minutes. Finally, the magnesium stearate is added through a sieve of 0.8mm width and, after mixing for a further 3 minutes, the mixture isintroduced in portions of 140 mg each into size 0 (elongated) gelatindry-fill capsules.

Example 23

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

1-oxo-2-(2,6-dioxo- 5.0 g piperidin-3-yl)-7-amino- isoindoline sodiumchloride 22.5 g phosphate buffer pH 7.4 300.0 g demineralized water to2500.0 mL

1-Oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline is dissolved in1000 mL of water and filtered through a microfilter. The buffer solutionis added and the whole is made up to 2500 mL with water. To preparedosage unit forms, portions of 1.0 or 2.5 mL each are introduced intoglass ampoules (each containing respectively 2.0 or 5.0 mg of imide).

Example 24

Tablets, each containing 50 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline, can beprepared in the following manner:

Constituents (for 1000 tablets) 1-oxo-2-(2,6-dioxo- 50.0 g piperidin-3-yl)-4,5,6,7- tetrafluoroisoindoline lactose 50.7 g  wheatstarch 7.5 g polyethylene glycol 6000 5.0 g talc 5.0 g magnesiumstearate 1.8 g demineralized water q.s.

The solid ingredients are first forced through, a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35° C., forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

Example 25

Tablets, each containing 100 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline, can beprepared in the following manner:

Constituents (for 1000 tablets) 1-oxo-2-(2,6-dioxopiperidin-3-yl)- 100.0g 4,5,6,7-tetrachloroisoindoline lactose 100.0 g wheat starch  47.0 gmagnesium stearate  3.0 g

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, lactose, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to 100 mL of boilingwater. The resulting paste is added to the pulverulent substances andthe mixture is granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 6 mm diameterwhich are concave on both sides.

Example 26

Tablets for chewing, each containing 75 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline, can beprepared in the following manner:

Composition (for 1000 tablets) 1-oxo-2-(2,6-dioxo- 75.0 gpiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline mannitol 230.0 g  lactose150.0 g  talc 21.0 g glycine 12.5 g stearic acid 10.0 g saccharin  1.5 g5% gelatin solution q.s.

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol and the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50° C. and again forced through a sieve of 1.7 mm mesh width.1-Oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline, theglycine and the saccharin are carefully mixed, the mannitol, the lactosegranulate, the stearic acid and the talc are added and the whole ismixed thoroughly and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking groove onthe upper side.

Example 27

Tablets, each containing 10 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline, can beprepared in the following manner:

Composition (for 1000 tablets) 1-oxo-2-(2,6-dioxo- 10.0 gpiperidin-3-yl)-4,5,6,7- tetramethylisoindoline lactose 328.5 g  cornstarch 17.5 g polyethylene glycol 6000  5.0 g talc 25.0 g magnesiumstearate  4.0 g demineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. Then the active imide ingredient, lactose, talc, magnesiumstearate and half of the starch are intimately mixed. The other half ofthe starch is suspended in 65 mL of water and this suspension is addedto a boiling solution of the polyethylene glycol in 260 mL of water. Theresulting paste is added to the pulverulent substances, and the whole ismixed and granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

Example 28

Gelatin dry-filled capsules, each containing 100 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline, canbe prepared in the following manner:

Composition (for 1000 capsules) 1-oxo-2-(2,6-dioxopiperidin-3- 100.0 g yl)-4,5,6,7-tetramethoxyisoindoline microcrystalline cellulose 30.0 g sodium lauryl sulfate 2.0 g magnesium stearate 8.0 g

The sodium lauryl sulfate is sieved into the1-oxo-2-(2.6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindolinethrough a sieve of 0.2 mm mesh width and the two components areintimately mixed for 10 minutes. The microcrystalline cellulose is thenadded through a sieve of 0.9 mm mesh width and the whole is againintimately mixed for 10 minutes. Finally, the magnesium stearate isadded through a sieve of 0.8 mm width and, after mixing for a further 3minutes, the mixture is introduced in portions of 140 mg each into size0 (elongated) gelatin dry-fill capsules.

Example 30

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

1-oxo-2-(2,6-dioxopiperidin-3-yl)- 5.0 g 4,5,6,7-tetrafluoroisoindolinesodium chloride 22.5 g phosphate buffer pH 7.4 300.0 g demineralizedwater to 2500.0 mL

1-Oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline isdissolved in 1000 mL of water and filtered through a microfilter. Thebuffer solution is added and the whole is made up to 2500 mL with water.To prepare dosage unit forms, portions of 1.0 or 2.5 mL each areintroduced into glass ampoules (each containing respectively 2.0 or 5.0mg of imide).

Example 31

Tablets, each containing 50 mg of1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,can be prepared in the following manner:

Constituents (for 1000 tablets) 1-oxo-2-(2,6-dioxo-3-methylpiperidin-50.0 g  3-yl)-4,5,6,7- tetrafluoroisoindoline lactose 50.7 g  wheatstarch 7.5 g polyethylene glycol 6000 5.0 g talc 5.0 g magnesiumstearate 1.8 g demineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35° C. forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

Example 32

Tablets, each containing 100 mg of1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline, can be prepared inthe following manner:

Constituents (for 1000 tablets) 1-oxo-2-(2,6-dioxopiperidin- 100.0 g3-yl)-4-aminoisoindoline lactose 100.0 g wheat starch  47.0 g magnesiumstearate  3.0 g

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, lactose, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to 100 mL of boilingwater. The resulting paste is added to the pulverulent substances andthe mixture is granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 6 mm diameterwhich are concave on both sides.

Example 33

Tablets for chewing, each containing 75 mg of2-(2,6-dioxo-3-methylpiperidin-3-yl)-4-aminophthalimide, can be preparedin the following manner:

Composition (for 1000 tablets) 2-(2,6-dioxo-3-methylpiperidin- 75.0 g3-yl)-4-aminophthalimide mannitol 230.0 g  lactose 150.0 g  talc 21.0 gglycine 12.5 g stearic acid 10.0 g saccharin  1.5 g 5% gelatin solutionq.s.

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol and the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50° C. and again forced through a sieve of 1.7 mm mesh width.2-(2,6-Dioxo-3-methylpiperidin-3-yl)-4-aminophthalimide, the glycine andthe saccharin are carefully mixed, the mannitol, the lactose granulate,the stearic acid and the talc are added and the whole is mixedthoroughly and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking groove onthe upper side.

Example 34

Tablets, each containing 10 mg of2-(2,6-dioxoethylpiperidin-3-yl)-4-aminophthalimide, can be prepared inthe following manner:

Composition (for 1000 tablets) 2-(2,6-dioxoethylpiperidin-3-yl)- 10.0 g4-aminophthalimide lactose 328.5 g  corn starch 17.5 g polyethyleneglycol 6000  5.0 g talc 25.0 g magnesium stearate  4.0 g demineralizedwater q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. Then the active imide ingredient, lactose, talc, magnesiumstearate and half of the starch are intimately mixed. The other half ofthe starch is suspended in 65 mL of water and this suspension is addedto a boiling solution of the polyethylene glycol in 260 mL of water. Theresulting paste is added to the pulverulent substances, and the whole ismixed and granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

Example 35

Gelatin dry-filled capsules, each containing 100 mg of1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,can be prepared in the following manner:

Composition (for 1000 capsules) 1-oxo-2-(2,6-dioxo-3- 100.0 g methylpiperidin-3-yl)-4,5,6,7- tetrafluoroisoindoline microcrystallinecellulose 30.0 g  sodium lauryl sulfate 2.0 g magnesium stearate 8.0 g

The sodium lauryl sulfate is sieved into the1-oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,5,6,7-tetrafluoroisoindolinethrough a sieve of 0.2 mm mesh width and the two components areintimately mixed for 10 minutes. The microcrystalline cellulose is thenadded through a sieve of 0.9 mm mesh width and the whole is againintimately mixed for 10 minutes. Finally, the magnesium stearate isadded through a sieve of 0.8 mm width and, after mixing for a further 3minutes, the mixture is introduced in portions of 140 mg each into size0 (elongated) gelatin dry-fill capsules.

Example 36

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

1-oxo-2-(2,6-dioxo-3-methylpiperidin- 5.0 g3-yl)-4,5,6,7-tetrafluoroisoindoline sodium chloride 22.5 g phosphatebuffer pH 7.4 300.0 g demineralized water to 2500.0 mL

1-Oxo-2-(2,6-dioxo-3-methylpiperidin-3-yl)-4,5,6,7-tetrafluoroisoindolineis dissolved in 1000 mL of water and filtered through a microfilter. Thebuffer solution is added and the whole is made up to 2500 mL with water.To prepare dosage unit forms, portions of 1.0 or 2.5 mL each areintroduced into glass ampoules (each containing respectively 2.0 or 5.0mg of imide).

1. A method of treating cachexia or graft v. Host disease comprisingadministering to a patient in need of such treatment an effective amountof 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline:

or a salt or stereoisomer thereof.
 2. The method of claim 1, wherein asubstantially chirally pure (S) or (R) isomer is administered.
 3. Themethod of claim 1, wherein1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline, or a salt orstereoisomer thereof, is administered orally or parenterally.
 4. Themethod of claim 1, wherein cachexia is treated.
 5. The method of claim1, wherein graft v. host disease is treated.